Because cytomegalovirus (CMV) may contribute to restenosis and atherosclerosis, and because smooth muscle cells (SMCs) are involved in these disease processes, we examined CMV/SMC interactions. Using confocal microscopy to identify a redox sensitive fluorescent marker, we found that CMV infection of SMCs generates intracellular reactive oxygen intermediates (ROIs). CMV also activated the cellular transcription factor NFkappaB, as demonstrated by increased NFkappaB binding to DNA (electrophoretic mobility shift assay). Antioxidants inhibited activation, suggesting a role of ROIs in CMV-induced NFkappaB activation. By using antioxidants to assess the role of ROIs in modulating virally-mediated effects, we also found CMV-induced ROIs: 1)are critical to the transactivation of the viral major immediate promoter (MIEP) by its immediate early protein IE72 (determined by cotransfection of an IE72 expression vector and a reporter gene downstream of tche MIEP); 2) are necessary for IE72 expression (determined by immunocytochemistry) and viral replication (determined by viral titer assay on indicator cells) following CMV infection of SMCs. We also demonstrated that aspirin (which has antioxidant and other activities) inhibits HCMV-induced ROS generation, HCMV-induced NFkappaB activation, IE72 expression, and HCMV replication. We further determined that CMV infection induces the prooxidant cyclooxygenase-2 (COX-2), by assaying for prostaglandin E2 [PGE2] release into the supernatant. We found expression of COX-2 protein (by Western blot) in infected cells but not in uninfected cells. Finally, we demonstrated that the immediate early viral proteins IE72 and IE84 transcriptionally activate the COX-2 promoter, by contransfecting smooth muscle cells with a COX-2 reporter gene and IE72 and IE84 expression vectors.